Mu, delta and kappa opioid receptors modulate nociceptive, emotional and addictive behaviors. An interesting but poorly explored aspect of opioid receptor function is their agonist-independent activity, which may operate in addition to the classically described endogenous peptidic tone. Indeed, the delta receptor was reported as the first example of a G protein coupled receptor (GPCR) showing measurable spontaneous activity. Here we propose to develop tools to explore and manipulate spontaneous activity of delta and mu receptors both in vitro and in vivo. In Aim 1 we propose to use a random mutagenesis strategy to identify a large number of Constitutively Active Mutant (CAM) delta receptors. Mutations will be analyzed by computer 3D-modeling and CAM receptors will be studied for basal and inverse agonist responses in several distinct signaling pathways. This aim will (i) identify molecular mechanisms of receptor activation without any preconceived structural hypothesis, (ii) define active state(s) of the receptor and (iii) provide mutant receptors as tools for the second aim of the proposal. In Aim 2 we will investigate the significance of spontaneous activity of delta receptors in vivo. We will express CAM delta and mu receptors in cell lines to search for inverse agonists and neutral antagonists to be administered in vivo. We will develop knock-in mice expressing a selected CAM delta receptor in place of the endogenous receptor by homologous recombination. We will compare pharmacological activities of a delta inverse agonist and neutral antagonist in behavioral models of anxiety both in wild-type and the knock-in mice. Other physiological responses or behaviors to delta and mu inverse agonists will be studied in conjunction with other components. This second aim will (i) clarify the putative physiological role of spontaneously active delta opioid receptors and (ii) indicate whether these inverse agonists at opioid receptors are of any therapeutical interest. Additionally, the experimental design and data from this study should be of general interest to GPCR research and will significantly enhance our knowledge of receptor constitutive activity.